Heart disease is the number 1 killer in the United States. The beat-to-beat function of heart is largely controlled by the cycling of calcium between the cytoplasm and sarcoplasmic reticulum (SR) of cardiomyocytes, the cells of heart muscle. Human phospholamban (PLB) is expressed in the SR membrane of cardiomyocytes as a 30 kDa homopentamer, where it regulates cellular calcium level by a mechanism that depends on its phosphorylation. The goal of our proposal is to understand at an atomic level how phosphorylation and de-phosphorylation affect the structure and function of the PLB pentamer. The proposal consists of four specific aims. (1) Determine by solution NMR the structure of dephosphorylated PLB pentamer in detergent micelles, and validate the structure in the lipid bilayer environment of small bicelles. (2) Delineate the structural changes in the PLB pentamer upon phosphorylation by orientation restraints derived from NMR dipolar couplings. This involves the development of a paramagnetic alignment system for accurate measurements of dipolar couplings for the PLB pentamer reconstituted in bicelles. (3) Characterize the inhibitory interaction between the PLB pentamer and the SR calcium pump (Ca2+ATPase) by NMR chemical shift perturbation, fluorescence energy transfer, and potentially crystallographic methods. (4) Investigate, using liposome assays and other electrophysiology techniques for channel conductance measurement, whether the PLB pentamer functions as an ion channel, in addition to its established role as a regulator of the SR calcium pumps. The experiments are designed to answer the following questions: what is the ion permeability and selectivity of the PLB channel and how are they changed upon phosphorylation. Inadequate calcium cycling in cardiomyocytes leads to severe deterioration of heart function. A thorough understanding of PLB structure and mechanism will offer new opportunities for novel cardiac therapy, as it will allow us to rationally design methods for fine-tuning calcium cycling through the actions of PLB.
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